Mixed‐mode hydrophilic interaction/cation‐exchange chromatography (HILIC/CEX) of peptides and proteins

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Abstract
This review represents a summary of the development and application of a novel mixed‐mode HPLC approach to the separation and analysis of peptides and proteins termed hydrophilic interaction/cation‐exchange chromatography (HILIC/CEX). This approach combines the most advantageous aspects of two widely different separation mechanisms, i.e. a separation based on hydrophilicity/hydrophobicity differences between polypeptides overlaid on a separation based on net charge. Applications described include HILIC/CEX separations of cyclic peptides, α‐helical peptides, random coil peptides and modified or deletion products of synthetic peptides. In addition, the excellent resolving ability of HILIC/CEX for modified histone proteins is described. This approach is shown to represent an excellent complement to RP chromatography (RPC), as well as being a potent analytical tool in its own right.